G-protein coupled receptors (GPCRs) constitute a large protein family of receptors that sense molecules outside the cell and activate inside signal transduction pathways and, ultimately, cellular responses. Ligands that bind and activate these receptors include light-sensitive compounds, odors, pheromones, hormones, and neurotransmitters, and vary in size from small molecules to peptides to large proteins. GPCRs are integral to a number of disease processes, and are also the target of approximately 40% of all modern medicinal drugs. Despite their critical importance, current understanding of the structure and function of GPCRs is inadequate because of their low natural abundance. Thus, for structural studies, which require milligram quantities of purified membrane protein, production in heterologous systems is required, but has been extremely difficult to accomplish.
Existing GPCR-stabilizing processes are focused on preserving the GPCR once it has been dissolved in detergents. Other GPCR-stabilizing processes require addition of antagonist/agonists that force the GPCR into a more stable inactive state, or require addition of stabilizing lipid-like substances, which requires site-directed mutagenesis of the GPCR to generate a more stable structure. For instance, GPCRs may be separated and purified from different raw materials using affinity chromatography by means of a general affinity tag, such as a poly-histidine tag, or using a subsequent receptor-specific ligand column. If suitable immobilized ligands are unavailable, size-exclusion chromatography or other techniques may be applied.
As another example, CN102558344A relates to a method for separating and purifying GPCRs. The method is characterized in that the GPCRs are separated and purified from different raw materials by an ultrafiltration technology in a key process. US2006/0188964 provides a method for identifying a reagent in which a membrane protein is likely to crystallize, and a method for producing crystals of a protein which, in a cell, is a membrane-bound protein. U.S. Pat. No. 6,528,271 discloses a method of screening a compound for activity in controlling pain. The method comprises determining whether or not the compound inhibits β-arrestin binding to phosphorylated μ-opioid receptor.
The present invention features methods of stabilizing detergent solubilized GPCRs in powdered form, which can be stored at a temperature of −20° C. or lower and for a duration of 365 days or longer without substantial loss of photochemical functionality of the GPCRs after rehydration as compared to their pre-frozen state. None of the above references teaches a dry powdered form of GPCRs as the final product as disclosed in the presently claimed invention.
Any feature or combination of features described herein are included within the scope of the present invention provided that the features included in any such combination are not mutually inconsistent as will be apparent from the context, this specification, and the knowledge of one of ordinary skill in the art. Additional advantages and aspects of the present invention are apparent in the following detailed description and claims.